The overall objective of the proposed research is to elucidate the mechanism of protein biosynthesis in the mitochondria of animal cells and to compare this system to prokaryotic and eukaryotic cytoplasmic translational systems. During the past year, we have detected and partially purified the factor responsible for the binding of aminoacyltRNA to mitochondrial ribosomes (mtEF- Tu). We plan to complete the purification of this factor and to carry out a detailed characterization of its mechanism of action. In particular, we will examine the interaction mtEF-Tu with aminoacyltRNA, guanine nucleotides and ribosomes and its sensitivity to specific antibiotics. In addition, we will seek to determine whether a separate factor (analogous to the bacterial factor EFTs) is required to facilitate guanine nucleotide exchange during the recycling of mtEFTu. We have recently detected and partially purified the factor that catalyzes the translocation step of protein synthesis in mitochondria (mtEF-G). We plan to complete the purification of mtEFG and to examine its properties in detail. In this aspect of the proposed research, we hope to define the nature of the elongation cycle in mitochondria and to understand its relationship to the corresponding process in prokaryotes and in the eukaryotic cell cytoplasm. The second major focus of the proposed research is to begin an investigation into the process of protein chain initiation in animal mitochondria. In order to carry out this objective, we will seek to identify the factors required for chain initiation, examine their properties and define their roles in the initiation process. We will seek to determine how the 5' end of the mRNA is recognized as the initiation signal on mitochondrial messenger RNAs. The results obtained will permit us to compare the process of protein chain initiation in this essential cell organelle to other cellular and organelle systems. A longterm objective is to develop a defined in vitro protein synthesizing system from animal cell mitochondria in which to study the regulation of organelle protein synthesis and its integration into the complex metabolism of the eukaryotic cell.